Potentiometric Transducers for the Selective Recognition of Risperidone Based on Molecularly Imprinted Polymer

Graphite Coated Electrodes (GCE) based on molecularly imprinted polymers were fabricated for the selective potentiometric determination of Risperidone (Ris). The molecularly imprinted (MIP) and nonimprinted (NIP) polymers were synthesized by bulk polymerization using (Ris.) as a template, acrylic acid (AA) and acrylamide (AAm) as monomers, ethylene glycol dimethacrylate (EGDMA) as a cross-linker and benzoyl peroxide (BPO) as an initiator. The imprinted membranes and the nonimprinted membranes were prepared using dioctyl phthalate (DOP) and Dibutylphthalate (DBP) as plasticizers in PVC matrix. The membranes were coated on graphite electrodes. The MIP electrodes using (AA) and (AAm) showed a near nernstian and nernstian response with slopes of 55.2±0.1 and 59.0±0.2 mV/decade, correlation coefficient (r 2 ) 0.9997 and 0.9999, a linear response for a concentration range of (1.0×10 -6 1.0×10 -2 ) M and (5.0x 10 -7 to 1.0 x 10 -2 ) M respectively. The response time of the prepared electrodes was less than 30 seconds. The electrode responses were stable in a pH range (4-8). The electrodes exhibited good selectivity over a wide range of interference. The most effective electrode (Ris-MIP GCE) was used to determine the concentration of (Ris.) in some pharmaceutical formulations. The electrodes could be successfully used within (7and13) weeks respectively without any drift.


Introduction
Risperidone (Ris.)belongs as a chemical structure to an important antipsychotic medication chemical group called "benzioxazoles" (1,2) that is mainly used to treat schizophrenia , schizoaffective disorder, the mixed and manic states of bipolar disorder, and irritability in people with autism.(Ris.)undergoes hepatic metabolism and renal excretion. (2)It is a second-generation atypical antipsychotic agents (SGAs) which are broadspectrum antagonists of dopamine, alpha noradrenergic, serotonin and histamine receptors.The structural formula of (Ris.) is shown in Scheme (1).

Scheme (1): The Structural Formula of Risperidone.
The mechanism by which atypical drugs achieve this highly selective approach to treatment is the antagonism of a specific serotonin receptor, 5-HT 2A .This receptor can be found on the axon terminal of neurons that produce dopamine.When serotonin activates those receptors, the release of dopamine decreases.By building into the typical antipsychotics a mechanism to block the 5-HT 2A receptors from serotonin, dopamine release is increased (3,4) .
Molecular imprinting is a process in which functional and cross-linking monomers are copolymerized in the presence of the target analyte (the imprint molecule), which acts as a molecular template. (8,9) he functional monomer initially interacts with the imprint molecule to form a multi-action spot (complex). (10)After polymerization, their functional groups are held in position by the highly cross-linked polymeric structure (8,9) .Additionally, the steric arrangement of these interactions around a given substrate (molecular template) is a crucial aspect necessary for the creation of binding pockets providing complementary size, shape, and functionality for preferentially facilitating selective recognition along with a high affinity toward the target.Thus, the recognition process in MIPs may be described in analogy with mechanisms established for enzymesubstrate-complexes such as the lock-and-key (11) .
The synthesis of MIPs usually involves a parallel process involving synthesis of a control non-imprinted polymer (NIP) under conditions identical to those of the MIP except that the template is absent.In principle, the NIP is entirely analogous to the MIP except that any binding sites within its porous structure are non-selective.The NIP can therefore be used as a benchmark for assessing the selectivity of the MIP (12) .

Apparatus
The FTIR spectra of MIPs and NIPs were obtained using FTIR-8400S spectrometer (Shimadzu, Japan).A Shimadzu 1800 UV-Visible, double beam spectrophotometer (Kyoto-Japan) with 1 cm path length quartz cell.All potentiometric measurements were made at room temperature with a pH/mV meter Philips pW9421, England.The potentiometric measurements were recorded using the fabricated Ris-MIP membrane coated on a homemade graphite electrode in conjugation with a reference saturated calomel electrode (SCE).The pH values were recorded using WTW GmbH , inolab pH 7110 Germany pH meter.

Synthesis of Ris-MIPs
MIPs for (Ris.) were prepared using a bulk polymerization procedure.3.6542 mmol of the template (Ris) was dissolved in 5 mL of chloroform in a thick walled glass tube.14.6168 mmol of functional monomer acidic (AA) or basic (AAm), 73.084 mmol of crosslinker (EGDMA), and 0.32 mmol of initiator (BPO) were then added to the above solution respectively.The mixture was degassed by purging nitrogen for 10 minutes and shacked in ultrasonic water bath.The reaction flask was then removed, sealed and heated at 70°C in a water bath for 15 min.to allow initiation of the reaction.Orange colored polymers with a rigid structure were formed.Schemes ( 2) and ( 3) illustrate the synthesis of MIPs for (Ris.)based on (AA) and (AAm) as acidic and basic monomers.
Non-reacted species (excessive reagents or template) were removed from the polymers by consecutive washout of the particles with chloroform then diethyl ether and were dried at room temperature overnight.The polymers were then crushed grounded using mortar and pestle and sieved to particles size 150μm (using 100 mesh sieve).The template (Ris.) was removed by repeated washing the MIPs successively with 100 mL portions of 30% (v/v) acetic acid / water solution.The elimination of (Ris.) from the MIPs was confirmed by measuring the absorbance of the washout solution at 274 nm.After the polymer was completely dried at ambient temperature, it was used as an active material in the selective sensor.A control non-imprinted polymer (NIP) was prepared according to the same procedure, but excluding the target molecule, (Ris.) (13,14)

Construction of the MIP membrane graphite electrode
The sensing PVC membrane was prepared by mixing 0.17 g of high molecular weight PVC, 0.4 g of plasticizer either DOP or DBP and 0.02 g of the MIP or NIP particles.After homogenization, 2-3 mL of THF was added and stirred.The graphite coated electrode (GCE) was constructed by dipping a graphite rod (3 mm diameter and 10 cm long) in the above solution and allowing it to dry in air.The procedure was repeated until the coating thickness is about 1.0 (±0.1) mm. (13,15)

EMF measurements
The performance of the electrode was investigated by measuring the e.m.f.using the following cell:

ʼs stock solution
A standard stock solution of (Ris.) was prepared by dissolving accurately weighed 0.4105 g of pure drug in 10 mL of 0.1 M HCl and the volume was completed up to the mark in 100 mL volumetric flask with distilled water.Working solutions were freshly prepared by subsequent dilutions.

Potentiometric measurements of the proposed sensors
All potentiometric measurements were carried out at room temperature (25±2˚C) in stirred solutions.Aliquots from standard drug solution 1.0×10 −2 M were diluted to obtain a series of solutions with concentrations ranged (5.0×10 -9 -1.0×10 -2 ) M in 50mL volumetric flasks.30mL of each solution was transferred to a beaker and the e.m.f.values were recorded using (Ris.)electrode in conjunction with a reference SCE after stabilization to ±0.2 mV and the calibration curve was plotted.The calibration plot was used for measuring unknown concentrations under the same conditions. (16)

Binding experiments
Binding experiments were carried out by placing 0.02 g of Ris-MIP in contact with 10.0mL (Ris.)solutions ranged (0.005-0.32) mM .The mixtures were shacked for 2h at (25˚C±2) and the solid phase was separated by centrifugation 6000 ) rpm) for 40 minutes.The concentrations of free drugs in the supernatant were detected by UV-VIS spectrophotometry at 238 nm.The amount of drugs bound to the polymer was calculated by subtracting the concentration of free drug from the initial drug concentration.

Determination of (Ris.) in pharmaceutical formulations
MIP electrode for (Ris.)based on (AAm) as basic monomer was applied for the direct and standard addition determinations of (Ris.) in two pharmaceutical formulations; Respal® (2mg) caplets and Rispond (4mg) tablets.Ten tablets were finely ground, homogenized, then 0.2546 gm of Respal®, 0.3572gm of Respond were weighed accurately and separately, dissolved in 12mL of 0.1NHCl.The mixture was sonicated for 20min to aid dissolution and left to stand for (1 h).The solution was filtered by using Whatman filter paper No.41 to avoid any suspended or un-dissolved material before use.The resulting filtrate was transferred into a 25 ml volumetric flask and the volume was completed to the mark with distilled water to get 1.0×10 -3 M. Working solutions were freshly prepared by subsequent dilutions with distilled water, and analyzed by the recommended procedure.

FTIR of acidic and basic MIPs of (Ris.)
The FTIR spectra of (Ris.), control nonimprinted and molecularly imprinted polymer based on (AA) as a functional monomer prepared using radical bulk polymerization are shown in Figure (1).Both polymers have similar IR spectra indicating the similarity in the backbone structure.In the IR spectra, the absorptions due to sp 2 -CH str.(3228)cm -1 , sp 3 -CH str.2peaks(2985,2956) cm -1 , C=O str.(1732) cm -1 , C=C str.residual(1668) cm -1 , sp 3 -CH scissoring (1456) cm -1 , sp 3  In addition to backbone similarity concluding, those absorbances attributed to the previous groups for the molecularly imprinted polymer are relatively stronger than for non-imprinted polymer.From this comparison it was found that presence of imprint molecule (Ris.)causes incorporation of ethylene glycol dimethacrylate in the preparation of polymer to be increased. (17)On the other hand the FTIR spectra of (Ris.), control non-imprinted and molecularly imprinted polymer based on (AAm) as a functional monomer prepared using radical bulk polymerization are shown in Figure (2).Both polymers have similar IR spectra indicating the similarity in the backbone structure.In the IR spectra, the absorptions due to -NH2 str.2peaks (3435, 3370) ) cm -1 , sp 2 -CH str.( 3200) ) cm -1 , sp 3 -CH str.( 2957) ) cm -1 , Ester C=O str.( 1726) ) cm -1 , Amide C=O str.( 1690) ) cm -1 , C=C str.Residual(1645) ) cm -1 , NH ben.( 1535) ) cm -1 , sp 3 -CH scissoring(1456) ) cm -1 , sp 3 1051) ) cm -1 , sp 2 -CH ben.Out-of-plane(948) ) cm -1 were observed.In addition to backbone similarity concluding, those absorbances attributed to the previous groups for the molecularly imprinted polymer are relatively stronger than for non-imprinted polymer.From this comparison it was found that presence of imprint molecule (Ris.)causes incorporation of ethylene glycol dimethacrylate in the preparation of polymer to be increased (17) .
The life time of electrodes were estimated by their periodical recalibration and calculations of slope and limit of detection.A drift in the performance of electrodes I and III was noticed after 7 and 13 weeks respectively which indicate the end of electrodes lives.
Two membrane compositions were investigated by using two types of plasticizers (DOP and DBP) as shown in Figure (5).The results shown in table (1) for electrodes (III and V) indicate that membrane plasticized with (DOP) displayed better performance than that plasticized with (DBP).The (DOP) appeared to be more compatible with the Ris-MIP as it produces a homogenous and clear membrane having better slope and longer life time than (DBP).

Table (1):
The characteristics of the ( Ris.) GCE based on MIPs and NIPs using different functional monomers and plasticizers.

Effect of pH
The effect of pH on the potential readings of the electrodes (I and III) was studied by immersing a combined glass electrode, (Ris.)GCE and a saturated calomel reference electrode in 50 ml beakers containing 25 ml aliquots of 1.0×10-3 and 1.0×10-4 M of (Ris.)aqueous solutions.The pH of each solution was adjusted by 0.1N sodium hydroxide or hydrochloric acid solutions.The potential reading at each pH value ranging 1-10 was recorded.A plot of pH values versus the potential of the electrode is illustrated in figure (6).The electrode potential did not affect by the pH change in the range (4 -8) which considered as the working pH range of the proposed sensor.At pH lower than 4 the electrode potential decreased due to the formation of di-protonated form of (Ris.)where nitrogen atom of the pyrimidine ring is the preferred site for the second protonation (19).At pH value higher than 8, the decreased electrode potential might attribute to the deprotonation form of (Ris.) which lead to the precipitation of the drug in the test solution.

The selectivity study
The influence of the interfering ions on the response behavior of ion selective membrane electrode is usually described in terms of selectivity coefficients.The selectivity coefficients for (Ris.) over a wide range of interfering molecules, monovalente and di-valent ions were determined by the separate solutions method (SSM) using electrode (III) and listed in table (2).The selectivity coefficients calculated for (Ris.) over each interference at a A = a B in a series of concentrations ranged (1.0×10 -7 -1.0×10 -2 ) M for each of the two separate solutions ,i.e. the (Ris.)solutions and the interference solutions, were below the unity which indicates that these species do not interfere in the determination of (Ris.) .
The values of the selectivity coefficients were calculated according to the following equation (20) : Plots of selectivity of electrode (III) to interfering molecules and ions are shown in figure ( 7)

Validation of the proposed method
The proposed sensor no.(III) was successfully applied for the determination of (Ris.) in pure solutions.Four solutions of (Ris.) with different concentrations were prepared and analyzed with triplicate measurements.The validation of the method was achieved by the accuracy and precision test of the proposed sensor (III) expressed in terms of percent relative error and percent relative standard deviation for inter-day (repeated over 3 days) and intra-day (repeated independently three times a day) measurements of the prepared solutions.The results are shown in table (3).The recoveries of (Ris.) for each concentration were also calculated.The results have shown good accuracy and precision.

Determination of (Ris.) in pharmaceutical formulations
The direct, the standard addition (S.A) and multiple standard addition (M.S.A) methods were applied for the determination of (Ris.) in two types of pharmaceutical formulations; Rispal @ 2mg caplets and Rispond 4mg tablets.The results obtained using graphite coated electrode no.(III) are listed in Table (4).

Table (4):
Analysis of (Ris.) in some pharmaceutical formulations by the direct and the standard addition methods using electrode (III).

*Average of three determinations
Multiple standard addition (M.S.A) method has exhibited better results over the direct method.The method showed RSD% ranged between (0.0653-0.4965) and Rec% ranged between (99.0400 -101.1500).The reported percent recoveries of the drugs in Table 4 were calculated using the values reported by the manufacturer which was obtained using the recommended assay by the British Pharmacopoeia.

Binding characteristics
To understand the way how small molecules interact with adsorbent surface, the mode of binding and site distributions in the interaction, adsorption isotherms are an important tool obtained by plotting of the equilibrium concentrations of bound MIP versus free drug.In liquid-phase applications of MIPs, a molecule in solution interacts with binding sites in a solid adsorbent, and after equilibrium the free drug concentration becomes constant and is easily quantified to plot the corresponding adsorption isotherm. (21)his was determined by plotting the binding capacity (Q) against the free (Ris.).Q was calculated according to the following equation: (22) ( ) Where Q is the binding capacity of MIPs (µmol/g), C i is the initial (Ris.)concentration (mM), C free is the final (Ris) concentration (mM), V s is the volume of test solution (L) mass MIP is the mass of the dried MIP (g).The adsorption isotherms obtained after shaking varying concentrations of (Ris.) with the synthesized particles for 2 hours in a thermostatic water bath shaker at 25°C were plotted in Figs.(8).